Crystal structures of the SARS-CoV-2 nucleocapsid protein C-terminal domain and development of nucleocapsid-targeting nanobodies

The ongoing outbreak of COVID-19 caused by SARS-CoV-2 has resulted in a serious public health threat globally. Nucleocapsid protein is a major structural protein of SARS-CoV-2 that plays important roles in the viral RNA packing, replication, assembly, and infection. Here, we report two crystal structures of nucleocapsid protein C-terminal domain (CTD) at resolutions of 2.0 angstrom and 3.1 angstrom, respectively. These two structures, crystallized under different conditions, contain 2 and 12 CTDs in asymmetric unit, respectively. Interestingly, despite different crystal packing, both structures show a similar dimeric form as the smallest unit, consistent with its solution form measured by the size-exclusion chromatography, suggesting an important role of CTD in the dimerization of nucleocapsid proteins. By analyzing the surface charge distribution, we identified a stretch of positively charged residues between Lys257 and Arg262 that are involved in RNA-binding. Through screening a single-domain antibodies (sdAbs) library, we identified four sdAbs targeting different regions of nucleocapsid protein with high affinities that have future potential to be used in viral detection and therapeutic purposes.

Automated summary

The ongoing outbreak of COVID-19 caused by SARS-CoV-2 has posed a serious global public health threat. The crystal structures of the nucleocapsid protein C-terminal domain (CTD) have been reported, shedding light on the dimerization mechanism and RNA-binding site, and high-affinity sdAbs targeting different regions of the nucleocapsid protein have been identified, offering new possibilities for viral detection and therapeutic purposes.